We propose to develop an integrated sample processing and identification system to detect multidrug- resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis (MTB) directly from clinical sputum samples within 90-100 minutes. In the process, we seek to begin the transformation of the clinical mycobacteriology laboratory, by substituting most cultures with molecular techniques that are more rapid, sensitive and accurate. This proposal will leverage several successful programs in molecular diagnostics, sample processing, TB genetics and TB clinical trials at UMDNJ, the biotechnology company Cepheid, the NIH, and the International Tuberculosis Research Center in Masan, Korea to address the major public health threat of MDR and XDR MTB, category C select agents and emerging pathogens which kill many thousands of patients each year. Our partnership will develop a next generation integrated real-time PCR instrument and detection system that will enable us to detect the many targets and mutations required for MTB and mycobacteria other than tuberculosis (MOTT) detection and isoniazid (INH), rifampin (RIF) and fluoroquinolone (FQ) antibiotic resistance testing in a single real-time PCR tube. We will design and test new high Stoke's shift fluorophores and software which enable our GeneXpert sample processing and real-time PCR system to distinguish up to 15 different optical channels, and develop a new generation of "sloppy" molecular beacon probes that can test long sequences for multiple mutations. Not only will this effort result in a dramatically improved MTB assay, it will also produce a highly flexible instrument and assay system with broad utility in clinical diagnostics and biodefense. The specific aims of the project are to: 1) Develop mis- match tolerant or "sloppy" molecular beacons to identify mutations associated with INH and FQ resistance in nested PCR assays, and integrate these assays with MOTT and MTB detection and RIF resistance testing. 2) Develop a new "off axis" detection system to expand the 6-color detection range of the GeneXpert instrument. 3) Adapt the assay for the GeneXpert system creating reagent beads and new calibration methods. 4) To perform analytic and pre-clinical studies in preparation for large-scale clinical trials. RELEVANCE (See instructions): We will create a very simple to use detection system that can look for many different types of antibiotic resistances or detect many different types of DNA sequences at the same time. This system will be used to develop a test to rapidly detect severe forms of drug resistant Mycobacterium tuberculosis, permitting rapid quarantine of infectious patients and aiding in the selection of potentially life-saving treatment.